Abstract Specialized tissue-resident memory (TRM) T cells that persist at sites of infection are emerging as critical constituents of protective immunity. Designing vaccines that target the induction of lung TRM is a promising approach to improve protection against influenza, especially as T cells recognize peptide epitopes expressed across diverse viral strains, providing an avenue towards ?universal? protection. However, the transcriptional regulation controlling the transition of effector CD4 T cells to TRM is poorly understood, nor is it clear whether or not particular subsets of effector cells have a greater capacity to form TRM. This proposal will provide novel insight into the correlates of protective CD4 T cell responses against influenza, and produce high impact mechanistic data required to better elucidate the regulation of lung TRM generation. We recently developed an innovative murine influenza model in which the memory fate of virus-specific CD4 T cells can be restricted to only form highly functional and protective lung TRM. Our preliminary studies find dramatically increased expression of the transcription factor Eomesodermin (Eomes) in effectors that can only act as precursors for TRM versus in cells that can form circulating memory subsets. The role of Eomes during pathogen-specific CD4 T cell responses is not well-defined, but has been found to regulate aspects of cytokine production and cytotoxic programming in other animal models and human disease states. Through meta- analysis, we find unexpected overlap in gene signatures of cytolytic CD4 T cells and CD4 TRM, and that effectors with higher Eomes levels have increased cytotoxic capacity. We thus hypothesize that Eomes promotes a unique effector state in CD4 T cell effectors that is required to generate lung TRM and that is marked by a specialized functional repertoire including cytotoxicity. By modulating Eomes expression in CD4 T cells responding to influenza infection and intranasal vaccination, this proposal will provide clear and novel findings needed for innovative strategies to promote the most protective kinds of CD4 memory in the right locations. In Aim 1, we will determine the extent to which Eomes regulates cytokine production and cytotoxic potential in CD4 effectors, as well as how Eomes impacts transcriptional regulation during the effector to memory transition. We will also directly test the protective capacity of Eomes-deficient versus wild-type CD4 effectors. In Aim 2, we will determine how Eomes impacts the lung CD4 TRM landscape, and the extent to which hallmark protective TRM functions are modulated in Eomes-deficient cells. Finally, we will whether high Eomes expression in effector cells acts as a rheostat to limit their ability to give rise to circulating memory cells.